1. Field of the Invention
The present invention is related to a high throughput screening method for compounds that impact taste. More specifically, the present invention relates to a screening method useful in the identification of compounds that affect taste sensation by modulating the activity of the ion channel TRPM5. The screening method, using fluorescent membrane potential dyes, allows for the rapid screening of thousands of compounds by providing a visual fluorescent readout that can be easily automated.
2. Background
Taste perception not only plays a critical role in the nutritional status of human beings, but is also essential for the survival of both lower and higher animals (Margolskee, R. F. J. Biol. Chem. 277:1-4 (2002); Avenet, P. and Lindemann, B. J. Membrane Biol. 112:1-8 (1989)). Taste perception is carried out by taste receptor cells (TRCs). TRCs perceive the multitude of compounds that are associated with a given taste, and convert that perception to a signal deciphered by the brain, resulting in sweet, bitter, sour, salty, or umami (savory) taste.
TRCs are polarized epithelial cells, meaning they have specialized apical and basolateral membranes. Taste buds contain 60-100 TRCs, each having a tiny portion of its membrane exposed on the mucosal surface of the tongue (Kinnamon, S. C. TINS 11:491-496 (1988)). Sensory transduction is initiated by sapid molecules, or “tastants,” that interact with microvillar processes on the apical membrane of TRCs. The tastants bind specific membrane receptors, leading to a voltage change across the cell membrane; in turn this depolarizes, or changes the electric potential of the cell, causing transmitter release and excitation of primary gustatory nerve fibers.
Ion channels are transmembrane proteins that form pores in a membrane and allow ions to pass from one side to the other (reviewed in B. Hille (Ed), 1992, Ionic Channels of Excitable Membranes 2nd ed., Sinauer, Sunderland, Mass.). Although certain ion channels are open under all physiological membrane conditions (so-called leaky channels), many channels have “gates” that open in response to a specific stimulus. As examples, voltage-gated channels respond to a change in the electric potential across the membrane, mechanically-gated channels respond to mechanical stimulation of the membrane, and ligand-gated channels respond to the binding of specific molecules. Various ligand-gated channels can open in response to extracellular factors, such as a neurotransmitters (transmitter-gated channels), or intracellular factors, such as ions (ion-gated channels), or nucleotides (nucleotide-gated channels). Still other ion channels are modulated by interactions with proteins, such as G-proteins (G-protein coupled receptors or GPCRs).
Most ion channel proteins mediate the permeation of one predominant ionic species. For example, sodium (Na+), potassium (K+), chloride (Cl−), and calcium (Ca2+) channels have been identified.
One recently discovered ion channel, TRPM5, has been shown to be essential for taste transduction. Perez et al., Nature Neuroscience 5:1169-1176 (2002); Zhang et al., Cell 112:293-301 (2003). TRPM5 is a member of the transient receptor potential (TRP) family of ion channels. TRPM5 forms a channel through the membrane of the taste receptor cell, and is believed to be activated by stimulation of a receptor pathway coupled to phospholipase C and by IP3-mediated Ca2+ release. The opening of this channel is dependent on a rise in Ca2+ levels. Hofmann et al., Current Biol. 13:1153-1158 (2003). The activation of this channel leads to depolarization of the TRC, which in turn leads to transmitter release and excitation of primary gustatory nerve fibers.
Because TRPM5 is a necessary part of the taste-perception machinery, its inhibition prevents an animal from sensing particular tastes. Although taste perception is a vital function, the inhibition, or masking, of undesirable tastes is beneficial under certain circumstances. For example, many active pharmaceutical ingredients of medicines produce undesirable tastes, such as a bitter taste. Inhibition of the bitter taste produced by the medicine may lead to improved acceptance by the patient. In other circumstances, enhancement of taste may be desirable as in the case of developing improved artificial sweeteners or in treatment of taste losses in groups such as the elderly. Mojet et al., Chem Senses 26:845-60 (2001).
TRPM5 displays voltage modulation and rapid activation/deactivation (“opening and closing”) kinetics upon receptor stimulation (Hofmann et al. 2003) which allows for the passage of monovalent cations, such as sodium and potassium. A closely related protein, TRPM4b, also shows Ca2+ dependent voltage modulation, but opens and closes much slower than TRPM5. Thus, TRPM5 is the first example of a voltage-modulated, Ca2+-activated, monovalent cation channel that has rapid activation/deactivation kinetics (Hofmann et al. 2003).
Ion channel activation or inhibition may be determined by measuring changes in cell membrane potential when cells are exposed to certain stimuli. This is an indirect method of evaluating ion channel modulation, as cell membrane potential may be affected by multiple channels.
One method for testing ion channel activity is to measure changes in cell membrane potential using the patch-clamp technique. (Hamill et al., Nature 294:462-4 (1981)). In this technique, a cell is attached to an electrode containing a micropipette tip which directly measures the electrical conditions of the cell. This allows detailed biophysical characterization of changes in membrane potential in response to various stimuli. Thus, the patch-clamp technique can be used as a screening tool to identify compounds that modulate activity of ion channels. However, this technique is difficult to master and requires significant expertise to generate consistent, reliable data. Moreover, this technique is time consuming and would allow fewer than two or three compounds per day to be screened for activity.
Ideally, methods of screening test compounds are high throughput (i.e., allow for many compounds to be screened quickly), automated, easy to use, sensitive, and selective. Screening assays should also provide a high signal to background noise ratio. (Baxter et al., J. Biomol. Screen. 7:79-85 (2002)). Background noise is the minimal stimulation that a compound produces regardless of its effect on the ion channel. The high ratio makes visualization of positive or negative modulators simpler because the smallest response will be seen over the background measurements. This leads to a clear identification of modulating compounds.
A potential high throughput method for determining ion channel modulation utilizes fluorescent dyes that produce a fluorescent signal when the cell membrane potential changes. Increases in fluorescence occur, because upon a change in the membrane potential, the fluorescent dyes “flip” their orientation in the cell membrane bilayer from an intracellular to extracellular location. This flip causes an increase in fluorescence that is easily detected and quantified usually using an optical reader. Optical readouts of ion channel function are favorable for high throughput screening because they are potentially sensitive, versatile, and amenable to miniaturization and automation. Present day optical readers detect fluorescence from multiple samples in a short time and can be automated. Fluorescence readouts are used widely both to monitor intracellular ion concentrations and to measure membrane potentials.
In an attempt to overcome some of the shortcomings of traditional fluorescent dyes, modified bisoxonol fluorescent dyes such as the FLIPR® Membrane Potential dyes (FMP) from Molecular Devices were developed.
FMP dyes have been effective in correlating fluorescence with membrane potential determined directly by patch-clamp recording for “slow” ion channels (Baxter et al., J. Biomol. Screen. 7:79-85 (2002); Behrendt et al., British J. Pharmacol. 141:737-745 (2004); and Whiteaker et al., J. Biomol. Screen. 6:305-312 (2001).
A major challenge in designing a high throughput screening (HTS) method for compounds that modulate a specific ion channel is that methods of determining channel activation are indirect. To identify compounds that affect taste through modulation of TRPM5 activity, there must be a demonstration that the effect of the compounds on taste is specific to TRPM5 and not also to one or more of the multitude of other channels and receptors located on the cell surface. Additionally, since TRPM5 activation is calcium dependent, specificity of the TRPM5/test compound interaction must be confirmed by excluding those compounds that also modulate GPCR-agonist calcium flux.
Therefore, there exists a need in the art for HTS assays that can distinguish compounds that modulate taste by specifically acting on TRPM5, from compounds that may act by other mechanisms and that may not affect taste perception. The claimed invention provides HTS methods that give rapid and specific results, have a high signal to background ratio, and are easy to use.